With the recent explosion in gene identification, it has become crucial to develop efficient tools for functional genomics. One of the most valuable is the use of antisense oligonucleotide technology to validate gene function in cell-based assays. The ability of antisense oligonucleotides to decrease cellular message levels is now well established. However, their efficacy depends, in part, on the cellular concentration achieved and on the location of the oligonucleotides within the cell (Garcia-Chaumont, 2000; Marcusson, 1998). Many agents have been developed for delivery of DNA, and several of these have been shown to deliver nucleic acids into cells in vitro. These agents include cationic polymers, such as polylysine, and cationic lipids. For example, the liposomal composition Lipofectin® (Felgner et al., 1987), containing the cationic lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and the neutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine), is widely used. See also Benimetskaya, 1998; Bennett, 1992; Cao, 2000; DeLong, 1999; Kang, 1999; Morris, 1997; Lewis, 1996; and Yoo, 2000.
However, little attention has been placed on the development of carriers optimized for oligonucleotides. This point is especially important since antisense oligonucleotides have become an integral part of functional genomics and target validation in drug discovery. Ideally, transfection agents should be easy to use and give reproducible, efficient transfection of oligonucleotides into cells with minimal interference with biological systems. Unfortunately, many known transfection agents suffer from problems such as poor functional delivery, cellular toxicity, or incompatibility with serum in the transfection medium.
Toxicity and/or inefficient delivery by such vehicles in a growing number of cell lines requires that new candidate delivery vehicles be prepared and tested for their activity, both in general and for cell specific delivery. However, polycationic carriers such as those known in the art typically must be synthesized, purified, and tested individually, and many cationic lipids require formulation with DOPE for optimal activity. Accordingly, they are not amenable to structural variation by combinatorial synthesis or to high throughput screening. To date, screening of such compounds has been carried out on limited numbers of known, preselected compositions. See, for example, Byk et al., 1998; van de Wetering et al., 1999. Accordingly, there is a need for more efficient, high throughput synthesis and screening of candidate transfection agents.
Lipid-cationic peptoid conjugates, referred to as “lipitoids” and “cholesteroids”, have been shown to be effective reagents for the delivery of plasmid DNA to cells in vitro. These agents are able to condense plasmid DNA into small particles, protect it from nuclease degradation, and efficiently mediate the transfection of several cell lines (Murphy, 1998). See, for example, co-owned PCT publications WO 98/06437 and WO 99/08711 (Zuckermann et al.), corresponding to co-owned U.S. applications Ser. Nos.08/910,647 and 09/132,808, which are hereby incorporated by reference. Complexing of lipid-peptoid conjugates with plasmid DNA is described in Huang et al., 1998. Such compounds have also been shown to efficiently deliver oligonucleotides (i.e., shorter-length DNA or DNA analogs) into a variety of primary and tumor cell lines, as described in co-owned and co-pending U.S. application Ser. No.09/648,254. This is in contrast to many commercially available transfecting agents, which are less effective in delivery of oligonucleotides than in delivery of plasmid DNA. The lipid-peptoid conjugates can be synthesized by automated synthesis on solid phase and do not need to be formulated with other lipids before use. Accordingly, such compounds are well suited for combinatorial synthesis and high throughput screening, as further described herein.